The Ras family of small guanine-nucleotide binding proteins plays a pivotal role in many intracellular signal transduction pathways, including those which regulate cellular growth and differentiation and those which contribute to cell activation (Bourne et al., Nature 348:125-132 (1990); Marshall, FASEB J. 9:1311-1318 (1995)). Moreover, many different receptors expressed on the surface of diverse cell types can result in the activation of signal transduction pathways that are importantly influenced by Ras, and these pathways in turn determine whether, and to what extent, these cells respond to such cell surface receptor-dependent activation by proliferating, differentiating (i.e., developing new functional characteristics), and/or expressing specific functions (FIG. 1).
Depending on the circumstances, these "down-stream" consequences of the activation of Ras-dependent signal transduction pathways can have either adaptive (physiological) or maladaptive (pathological) consequences. For example, controlled Ras-dependent cellular proliferation is required for wound healing, whereas poorly regulated Ras-dependent cellular proliferation can result in the development of cancer and other neoplasms. Similarly, appropriate Ras-dependent cell secretion of histamine, serotonin, cytokines and other mediators can be important for host defense against parasites and other pathogens, whereas the inappropriate activation of these same pathways, for example, by a reaction to a bee-sting in patients who are allergic to components of bee venom, can lead to fatal anaphylaxis. Thus, Ras represents a major regulator of many of the most fundamental biological processes involved in both health and disease.
The mechanism by which Ras regulates such processes, through interactions with other intracellular molecules, is quite complex. Ras proteins are membrane-associated proteins that cycle between an active GTP-bound form and an inactive GDP-bound form. As illustrated in FIG. 1, evidence is accumulating for the existence of many different classes of positive or negative regulators of Ras and positive or negative Ras signaling effectors, all of which, by definition, are thought to interact directly with the active GTP-bound form of Ras to influence cellular signaling for growth, differentiation and expression of function (Boguski and McCormick, Nature 366:643-654 (1993); Marshall, FASEB J. 9:1311-1318 (1995); Marshall, Curr. Opin. Cell Biol 8:197-204 (1996)). For example, some of the best-characterized Ras regulators include the GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (Boguski and McCormick, Nature 366:643-654 (1993)). The GAPs represent a family of Ras-binding proteins which stimulate the intrinsic rate of Ras GTP hydrolysis and thus negatively regulate the Ras-induced signaling by accelerating the conversion of active GTP-bound form of Ras to the inactive GDP-bound form. Recent studies have identified several GAPs specific for Ras proteins, which include p120-Ras GAP, neurofibromin (the protein encoded by the neurofibromatosis type 1 (NF1) gene), Gap1, Ral-GDS, Rsbs 1, 2, and 4, Rin1, MEKK-1, and phosphatidylinositol-3-OH kinase (PI3K) (Boguski and McCormick, Nature 366:643-654 (1993)).
In contrast to Ras regulators, which function primarily by influencing the amount of Ras which is in the GTP-bound active, as opposed to the GDP-bound inactive, form, Ras effectors are thought to influence the ability of active, GTP-bound Ras to initiate signaling (FIG. 1). In the case of many Ras-interacting proteins which can influence the intensity of Ras-dependent signaling, it is not yet clear to what extent they function as effectors as opposed to regulators; such proteins can therefore be called Ras regulators/effectors (Boguski and McCormick, Nature 366:643-54 (1993); Han and Colicelli, Mol. Cell. Biol. 15:1318-1323 (1995); Marshall, FASEB J. 9:1311-1318 (1995); Marshall, Curr. Opin. Cell. Biol. 8:197-204 (1996)).
A large number of cell types demonstrate the importance of Ras in critical cell signaling events. For example, mast cells are important effector cells in IgE-dependent immune responses and allergic diseases (Galli, New Engl. J. Med. 328:257-265 (1993)), and mast cells also contribute to host defense against parasites and bacteria (Echtenacher et al., Nature 381:75-77 (1996); Malaviya et al., Nature 381:77-80 (1996); Galli and Wershil, Nature 381:21-22 (1996)). Mast cells reside in virtually all vascularized tissues and express on their surface the high affinity receptor for IgE (Fc.epsilon.RI). Aggregation of Fc.epsilon.RI in mast cells by the interaction of receptor-bound IgE with specific multivalent antigen triggers the functional activation of mast cells, which results in the release of a spectrum of biologically active mediators (Ravetch and Kinet, Ann. Rev. Immunol. 9:457-492 (1991); Galli, New Engl. J. Med. 328:257-265 (1993); Beaven and Metzger, Immunol. Today 14:222-226 (1993)). Thus, mast cells activated by Fc.epsilon.RI-dependent mechanisms undergo degranulation, resulting in the release of preformed mediators, such as serotonin (5-HT) and/or histamine, the metabolism of arachidonic acid, leading to the release of newly synthesized lipid mediators, and the transcription, translation and secretion of several cytokines (Gordon et al., Immunol. Today 11:458-464 (1990); Galli, New Engl. J. Med. 328:257-265 (1993); Paul et al., Adv. Immunol. 53:1-29 (1993)).
Knowledge of the signaling pathways which result in the Fc.epsilon.RI-dependent secretion of mast cell mediators is increasing. In mast cells and basophils, a type of circulating leukocyte which shates many biochemical and functional characteristics with mast cells (Galli, New Engl. J. Med. 328:257-265 (1993)), the Fc.epsilon.RI receptor is a tetrameric complex comprised of a single 45 kDa .alpha. chain, which binds the Fc portion of IgE, a single 30 kDa .beta. chain, and a homodimer of two 10 kDa .gamma. chains (Ravetch and Kinet, Ann. Rev. Immunol. 9:457-492 (1991); Beaven and Metzger, Immunol. Today 14:222-226 (1993)). The .beta. and .gamma. chains contain immunoreceptor tyrosine-based activation motifs (ITAM) which couple the receptor to the src family of protein tyrosine kinases (PTK) p56lyn and p72syk (Beaven and Baumgartner, Curr. Opin. Immunol. 8:766-772 (1996)). The Fc.epsilon.RI-dependent activation of these PTKs in turn activates various downstream effector pathways, including those involving PLC.gamma.1 and the MAP kinase pathway (Beaven and Baumgartner, Curr. Opin. Immunol. 8:766-772 (1996)).
Recent studies have shown that the crosslinking of Fc.epsilon.RI in mast cells by IgE and specific antigen also results in the activation of Ras and of the associated Shc-Grb2-Sos pathway, which precedes Ras activation, and that the activation of this pathway is dependent on Syk (Jabril-Cuenod et al., J. Biol. Chem. 271:16268-16272 (1996)). These results suggest that in Fc.epsilon.RI-activated mast cells, as in T cells and B cells (the major types of lymphocytes responsible for cellular and humoral immunity) which have been activated via the T cell receptor or B cell receptor, respectively, the Shc-Grb2-Sos pathway can activate the MAP kinase pathway via the activation of Ras.
An effector pathway of Ras mediated by the Raf-1/Erk-activating kinases (MEKs)/Erk-MAP kinases cascade has been well-characterized in numerous systems (Treisman, Curr. Opin. Cell Biol. 8:205-215 (1996)), and this Ras-mediated pathway is important in the mast cell activation and mediator secretion that is induced by IgE- and antigen-dependent aggregation of Fc.epsilon.RI in these cells (Tsai et al., Eur. J. Immunol. 23:3286-3291 (1993); Offermanns et al., J. Immunol. 152:250-261 (1994); Hirasawa et al., J. Immunol. 154:5391-5402 (1995)). In addition, recent studies with rat RBL2H3 mast cells have shown that the Fc.epsilon.RI induction of Ras activation leads to transcriptional activation mediated by the transcription factors Elk-1 and the nuclear factor of activated T cells (NFAT) (Turner and Cantrell, J. Exp. Med. 185:43-53 (1997)). These Ras-dependent signaling pathways in mast cells appear to be complex. Thus, activation of the Raf-1/MEK/Erk cascade appears to be necessary and sufficient for the activation of Elk-1 activity in mast cells. However, the induction of NFAT by Fc.epsilon.RI in mast cells is also mediated in part by Rac-1, which is a putative Ras effector and a member of the Rho family of GTP binding proteins (Turner and Cantrell, J. Exp. Med. 185:43-53 (1997)) . Thus, the specific signaling pathways involved in Fc.epsilon.RI-mediated mast cell activation are still unclear.